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A comparative PET imaging study with the reversible and irreversible EGFR tyrosine kinase inhibitors [(11)C]erlotinib and [(18)F]afatinib in lung cancer-bearing mice.

Slobbe P, Windhorst AD, Stigter-van Walsum M, Smit EF, Niessen HG, Solca F, Stehle G, van Dongen GA, Poot AJ - EJNMMI Res (2015)

Bottom Line: Additionally, the effect of drug efflux transporter permeability glycoprotein (P-gp) on the tumor uptake of tracers was explored by therapeutic blocking with tariquidar.Under P-gp blocking conditions, no significant changes in tumor-to-background ratio were observed; however, [(18)F]afatinib demonstrated better tumor retention in all xenograft models.TKI-PET provides a method to image sensitizing mutations and can be a valuable tool to compare the distinguished targeting properties of TKIs in vivo.

View Article: PubMed Central - PubMed

Affiliation: Department of Radiology and Nuclear Medicine, VU University Medical Center, De Boelelaan 1117, Amsterdam, 1081 HV The Netherlands ; Department of Otolaryngology/Head and Neck Surgery, VU University Medical Center, De Boelelaan 1117, Amsterdam, 1081 HV The Netherlands.

ABSTRACT

Background: Tyrosine kinase inhibitors (TKIs) have experienced a tremendous boost in the last decade, where more than 15 small molecule TKIs have been approved by the FDA. Unfortunately, despite their promising clinical successes, a large portion of patients remain unresponsive to these targeted drugs. For non-small cell lung cancer (NSCLC), the effectiveness of TKIs is dependent on the mutational status of epidermal growth factor receptor (EGFR). The exon 19 deletion as well as the L858R point mutation lead to excellent sensitivity to TKIs such as erlotinib and gefitinib; however, despite initial good response, most patients invariably develop resistance against these first-generation reversible TKIs, e.g., via T790M point mutation. Second-generation TKIs that irreversibly bind to EGFR wild-type and mutant isoforms have therefore been developed and one of these candidates, afatinib, has now reached the market. Whether irreversible TKIs differ from reversible TKIs in their in vivo tumor-targeting properties is, however, not known and is the subject of the present study.

Methods: Erlotinib was labeled with carbon-11 and afatinib with fluorine-18 without modifying the structure of these compounds. A preclinical positron emission tomography (PET) study was performed in mice bearing NSCLC xenografts with a representative panel of mutations: an EGFR-WT xenograft cell line (A549), an acquired treatment-resistant L858R/T790M mutant (H1975), and a treatment-sensitive exon 19 deleted mutant (HCC827). PET imaging was performed in these xenografts with both tracers. Additionally, the effect of drug efflux transporter permeability glycoprotein (P-gp) on the tumor uptake of tracers was explored by therapeutic blocking with tariquidar.

Results: Both tracers only demonstrated selective tumor uptake in the HCC827 xenograft line (tumor-to-background ratio, [(11)C]erlotinib 1.9 ± 0.5 and [(18)F]afatinib 2.3 ± 0.4), thereby showing the ability to distinguish sensitizing mutations in vivo. No major differences were observed in the kinetics of the reversible and the irreversible tracers in each of the xenograft models. Under P-gp blocking conditions, no significant changes in tumor-to-background ratio were observed; however, [(18)F]afatinib demonstrated better tumor retention in all xenograft models.

Conclusions: TKI-PET provides a method to image sensitizing mutations and can be a valuable tool to compare the distinguished targeting properties of TKIs in vivo.

No MeSH data available.


Related in: MedlinePlus

Radiosynthesis of [18F]6 and subsequent condensation towards [18F]afatinib. MeCN, acetonitrile; MeOH, methanol; DBU, 1,8-diazabicyclo[5.4.0]undec-7-een; NMP, N-methylpyrrolidone.
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Sch2: Radiosynthesis of [18F]6 and subsequent condensation towards [18F]afatinib. MeCN, acetonitrile; MeOH, methanol; DBU, 1,8-diazabicyclo[5.4.0]undec-7-een; NMP, N-methylpyrrolidone.

Mentions: [18F]afatinib [21] was synthesized as previously reported (Scheme 2). Briefly, cyclotron-produced [18F]fluoride was azeotropically dried with acetonitrile/water (9/1, v/v) containing K[2.2.2] (4,7,13,16,21,24-hexaoxa-1,10-diazabicyclo[8.8.8]hexacosane, 13.0 mg, 34.6 μmol) and potassium carbonate (2.0 mg, 15 μmol). To the dried residue was added a solution of 3-chloro-4-trimethylammonium-nitrobenzene triflate (4, 3.0 mg, 14 μmol) in acetonitrile (0.7 mL), and the mixture was allowed to react for 25 min at 40°C. After the reaction mixture was quenched with water (7 mL), 3-chloro-4-[18F]fluoronitrobenzene ([18F]5) was trapped on a tC18 Plus Sep-Pak, rinsed with water (10 mL), and subsequently eluted with MeOH (1.5 mL) into a screw cap reaction vessel containing palladium on activated carbon (10%, 3 mg) and sodium borohydride (10.0 mg, 264 μmol). The reduction was carried out for 7 min at 20°C upon which the reaction was quenched by the addition of concentrated hydrochloric acid (37%, 0.1 mL). The thus obtained mixture was passed through a syringe filter (Millex LCR PTFE 0.45 μM/25 mm; Millipore Corporation, Darmstadt, Germany) into a new screw cap reaction vessel. The volatiles were removed in vacuo and under a helium flow (100 mL/min) at elevated temperatures (90°C for 5 min and 120°C for 2 min) to obtain the dry 3-chloro-4-[18F]fluoroaniline-HCl salt ([18F]6). The product was dissolved in N-methylpyrrolidine (NMP, 0.5 mL) and to this solution was added a solution of (S,E)-4-(dimethylamino)-N-(4-oxo-7-((tetrahydrofuran-3-yl)oxy)-3,4-dihydroquinazolin-6-yl)but-2-enamide (7, 2 mg, 6 μmol), benzotriazole-1-yl-oxy-tris-(dimethylamino)-phosphonium hexafluorophosphate (BOP), 5.5 mg, 12 μmol, and 1,8-diazabicyclo[5.4.0]undec-7-een, 2.7 μL (DBU), 18 μmol, in anhydrous NMP (0.5 mL), which was dissolved 15 min prior to addition. The obtained mixture was heated to 120°C for 30 min after which it was cooled to 20°C. The product was isolated by semi-preparative HPLC, carried out on a Jasco PU-2089 pump equipped with a C18 Alltima column (Grace, 5 μm, 250 mm × 10 mm; Crawford Scientific, Columbia, MD, USA) using MeCN/H2O/DiPA (40:60:0.2, v/v/v) as eluent at a flow rate of 4 mL/min, a Jasco UV1575 UV detector (λ = 254 nm), and a custom-made radioactivity detector. Chromatograms were acquired using ChromNAV software (version 1.14.01, Jasco). The collected fraction of the preparative HPLC purification containing the product was diluted with 50 mL of water and the total mixture was passed over a tC18 Plus Sep-Pak cartridge. The cartridge was then washed with 20 mL of sterile water after which the product was eluted from the cartridge with 1.0 mL of sterile 96% ethanol. The ethanol was diluted to 10 vol.% with formulation solution (7.09 mM NaH2PO4 in 0.9% NaCl, w/v in water, pH 5.2), and the complete solution was filtered over a Millex-GV 0.22-μm filter into a sterile 20 mL capped vial. In this way, a final IV injectable solution was provided of 10% ethanol in saline (containing 7.09 mM NaH2PO4) containing [18F]afatinib obtained at >98% radiochemical purity, in a total synthesis time of less than 120 min (from end of isotope production), at a high specific activity (287 ± 63 GBq/μmol), and in 17.0% ± 2.5% yield (corrected for decay, up to 3.5 GBq isolated).Scheme 2


A comparative PET imaging study with the reversible and irreversible EGFR tyrosine kinase inhibitors [(11)C]erlotinib and [(18)F]afatinib in lung cancer-bearing mice.

Slobbe P, Windhorst AD, Stigter-van Walsum M, Smit EF, Niessen HG, Solca F, Stehle G, van Dongen GA, Poot AJ - EJNMMI Res (2015)

Radiosynthesis of [18F]6 and subsequent condensation towards [18F]afatinib. MeCN, acetonitrile; MeOH, methanol; DBU, 1,8-diazabicyclo[5.4.0]undec-7-een; NMP, N-methylpyrrolidone.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4385286&req=5

Sch2: Radiosynthesis of [18F]6 and subsequent condensation towards [18F]afatinib. MeCN, acetonitrile; MeOH, methanol; DBU, 1,8-diazabicyclo[5.4.0]undec-7-een; NMP, N-methylpyrrolidone.
Mentions: [18F]afatinib [21] was synthesized as previously reported (Scheme 2). Briefly, cyclotron-produced [18F]fluoride was azeotropically dried with acetonitrile/water (9/1, v/v) containing K[2.2.2] (4,7,13,16,21,24-hexaoxa-1,10-diazabicyclo[8.8.8]hexacosane, 13.0 mg, 34.6 μmol) and potassium carbonate (2.0 mg, 15 μmol). To the dried residue was added a solution of 3-chloro-4-trimethylammonium-nitrobenzene triflate (4, 3.0 mg, 14 μmol) in acetonitrile (0.7 mL), and the mixture was allowed to react for 25 min at 40°C. After the reaction mixture was quenched with water (7 mL), 3-chloro-4-[18F]fluoronitrobenzene ([18F]5) was trapped on a tC18 Plus Sep-Pak, rinsed with water (10 mL), and subsequently eluted with MeOH (1.5 mL) into a screw cap reaction vessel containing palladium on activated carbon (10%, 3 mg) and sodium borohydride (10.0 mg, 264 μmol). The reduction was carried out for 7 min at 20°C upon which the reaction was quenched by the addition of concentrated hydrochloric acid (37%, 0.1 mL). The thus obtained mixture was passed through a syringe filter (Millex LCR PTFE 0.45 μM/25 mm; Millipore Corporation, Darmstadt, Germany) into a new screw cap reaction vessel. The volatiles were removed in vacuo and under a helium flow (100 mL/min) at elevated temperatures (90°C for 5 min and 120°C for 2 min) to obtain the dry 3-chloro-4-[18F]fluoroaniline-HCl salt ([18F]6). The product was dissolved in N-methylpyrrolidine (NMP, 0.5 mL) and to this solution was added a solution of (S,E)-4-(dimethylamino)-N-(4-oxo-7-((tetrahydrofuran-3-yl)oxy)-3,4-dihydroquinazolin-6-yl)but-2-enamide (7, 2 mg, 6 μmol), benzotriazole-1-yl-oxy-tris-(dimethylamino)-phosphonium hexafluorophosphate (BOP), 5.5 mg, 12 μmol, and 1,8-diazabicyclo[5.4.0]undec-7-een, 2.7 μL (DBU), 18 μmol, in anhydrous NMP (0.5 mL), which was dissolved 15 min prior to addition. The obtained mixture was heated to 120°C for 30 min after which it was cooled to 20°C. The product was isolated by semi-preparative HPLC, carried out on a Jasco PU-2089 pump equipped with a C18 Alltima column (Grace, 5 μm, 250 mm × 10 mm; Crawford Scientific, Columbia, MD, USA) using MeCN/H2O/DiPA (40:60:0.2, v/v/v) as eluent at a flow rate of 4 mL/min, a Jasco UV1575 UV detector (λ = 254 nm), and a custom-made radioactivity detector. Chromatograms were acquired using ChromNAV software (version 1.14.01, Jasco). The collected fraction of the preparative HPLC purification containing the product was diluted with 50 mL of water and the total mixture was passed over a tC18 Plus Sep-Pak cartridge. The cartridge was then washed with 20 mL of sterile water after which the product was eluted from the cartridge with 1.0 mL of sterile 96% ethanol. The ethanol was diluted to 10 vol.% with formulation solution (7.09 mM NaH2PO4 in 0.9% NaCl, w/v in water, pH 5.2), and the complete solution was filtered over a Millex-GV 0.22-μm filter into a sterile 20 mL capped vial. In this way, a final IV injectable solution was provided of 10% ethanol in saline (containing 7.09 mM NaH2PO4) containing [18F]afatinib obtained at >98% radiochemical purity, in a total synthesis time of less than 120 min (from end of isotope production), at a high specific activity (287 ± 63 GBq/μmol), and in 17.0% ± 2.5% yield (corrected for decay, up to 3.5 GBq isolated).Scheme 2

Bottom Line: Additionally, the effect of drug efflux transporter permeability glycoprotein (P-gp) on the tumor uptake of tracers was explored by therapeutic blocking with tariquidar.Under P-gp blocking conditions, no significant changes in tumor-to-background ratio were observed; however, [(18)F]afatinib demonstrated better tumor retention in all xenograft models.TKI-PET provides a method to image sensitizing mutations and can be a valuable tool to compare the distinguished targeting properties of TKIs in vivo.

View Article: PubMed Central - PubMed

Affiliation: Department of Radiology and Nuclear Medicine, VU University Medical Center, De Boelelaan 1117, Amsterdam, 1081 HV The Netherlands ; Department of Otolaryngology/Head and Neck Surgery, VU University Medical Center, De Boelelaan 1117, Amsterdam, 1081 HV The Netherlands.

ABSTRACT

Background: Tyrosine kinase inhibitors (TKIs) have experienced a tremendous boost in the last decade, where more than 15 small molecule TKIs have been approved by the FDA. Unfortunately, despite their promising clinical successes, a large portion of patients remain unresponsive to these targeted drugs. For non-small cell lung cancer (NSCLC), the effectiveness of TKIs is dependent on the mutational status of epidermal growth factor receptor (EGFR). The exon 19 deletion as well as the L858R point mutation lead to excellent sensitivity to TKIs such as erlotinib and gefitinib; however, despite initial good response, most patients invariably develop resistance against these first-generation reversible TKIs, e.g., via T790M point mutation. Second-generation TKIs that irreversibly bind to EGFR wild-type and mutant isoforms have therefore been developed and one of these candidates, afatinib, has now reached the market. Whether irreversible TKIs differ from reversible TKIs in their in vivo tumor-targeting properties is, however, not known and is the subject of the present study.

Methods: Erlotinib was labeled with carbon-11 and afatinib with fluorine-18 without modifying the structure of these compounds. A preclinical positron emission tomography (PET) study was performed in mice bearing NSCLC xenografts with a representative panel of mutations: an EGFR-WT xenograft cell line (A549), an acquired treatment-resistant L858R/T790M mutant (H1975), and a treatment-sensitive exon 19 deleted mutant (HCC827). PET imaging was performed in these xenografts with both tracers. Additionally, the effect of drug efflux transporter permeability glycoprotein (P-gp) on the tumor uptake of tracers was explored by therapeutic blocking with tariquidar.

Results: Both tracers only demonstrated selective tumor uptake in the HCC827 xenograft line (tumor-to-background ratio, [(11)C]erlotinib 1.9 ± 0.5 and [(18)F]afatinib 2.3 ± 0.4), thereby showing the ability to distinguish sensitizing mutations in vivo. No major differences were observed in the kinetics of the reversible and the irreversible tracers in each of the xenograft models. Under P-gp blocking conditions, no significant changes in tumor-to-background ratio were observed; however, [(18)F]afatinib demonstrated better tumor retention in all xenograft models.

Conclusions: TKI-PET provides a method to image sensitizing mutations and can be a valuable tool to compare the distinguished targeting properties of TKIs in vivo.

No MeSH data available.


Related in: MedlinePlus